Useful substances produced by microbes have been widely used as industrial enzymes, antibiotics, etc. while 95-99% of the microbes present in the natural world are unable to be cultured and such microbes where culture is impossible have not been industrially utilized yet. However, useful substances such as novel industrial enzymes and antibiotics can be produced in large quantities if gene of industrially useful enzymes is obtained from microbes which are unable to be cultured or if biosynthetic gene of useful substances such as antibiotics produced by microbes and the said gene can be expressed in an appropriate host. Therefore, its usefulness in industry is quite significant.
Preparation of the above-mentioned useful gene from microbes usually starts from the procedure where the microbe having useful gene is isolated and cultured to extract DNA from the said microbe whereupon a gene library is prepared. Principally, the said gene library is introduced and expressed in an appropriate host and an active transformant is screened and selected by an appropriate method. However, in such a method, much time and labor are needed for the preparation of useful gene from the microbe which occupies only a part of a specimen where various microbes are mixed or from the microbe which is unable to be isolated and cultured. Especially when the amount of the microbe having useful gene in the mixture is small and further when the said microbe is unable to be cultured, preparation of the useful gene from the said microbe is substantially impossible. If the gene derived from the microbe which is present only in little amount in the gene specimen or derived from the microbe which is unable to be cultured in the specimen or, in other words, if the rare gene or the trace gene can be relatively concentrated, screening of the useful gene thereafter becomes efficient whereupon labor, time and cost therefor can be greatly saved.
In the meanwhile, it has been believed that cells constituting the human body are classified into 200 or more types, that each of the cells has common genome having about 100,000 types of genes and that several tens of thousands of genes are expressed depending upon the cell type. Investigation of expression of such gene is becoming more and more important not only for obtaining the information concerning the function of each gene but also for clarifying the life process. In addition, as a result of detailed analysis which has been conducted up to now for small amount of relatively limited gene, it has been found that plural genes act in a cooperative manner in many life processes.
Expression of gene is roughly classified into three classes according to the expressed amount. They are an abundant class of about 103-4 copies per cell, an intermediate class of about 102 copies and a rare class of only about 101 copies. On the other hand, with regard to the type of the expressed gene, there are as many as several tens of thousands types per cell in mammals and most of the genes belong to the rare class. Thus, with regard to expressed gene in the cell, there are only few types of gene of an abundant class having many expressed amount (103-4 copies) and there are quite a many types of gene of a rare class having only a little expression (101 copies) (e.g., Alberts, B., et al. (1989) Molecular Biology of the Cell, 2nd Edition, Garland Publishing Inc.). Under such circumstances, there has been a necessity for an art where many types of genes including rare gene is analyzed in more detail and, as a result of conducting such an analysis, its utilization in a medical field such as genetic diagnosis has been expected.
With regard to the analysis of rare gene at present, there has been known, for example, a method where a multiplex PCR is a base including a canonizing method (Minoru S. H. Ko (1990) Nucleic Acids Res., 18, 5705-5711), a differential display method (Liang, P., and Pardee, A. B. (1992), Science, 257, 967-971) and a molecular index method (Kikuya Kato (1995), Nucleic Acids Res., 23, 3685-3690, etc.) and also a method using DNA chips. In a canonizing method, a high-molecular nucleic acid mixture is placed under a hybridization condition and, after an appropriate period, nucleic acid becoming a double-stranded state is separated from that remaining in a single-stranded state whereupon the rare gene can be concentrated to the amount of the same degree as the abundant gene. In a canonizing method however, it does not happen that, after the treatment, numbers of rare gene are more than those of abundant gene and, therefore, the effect of concentration is limitative. In case an analysis using a multiplex PCR such as a differential display method is carried out, it has been known that a competitive PCR takes place and a strong bias is applied to the existing amount of the gene whereby the detecting sensitivity lowers as compared with the common PCR and accordingly that detection of gene of the rare class becomes difficult (David, J. Bertioli, et al. (1995) Nucleic Acids Res., 23, 4520-4523). As a method for overcoming such disadvantages, there is disclosed a method in Japanese Patent Laid-Open No. 2000/37,193 where known gene which is abundantly present in a nucleic acid sample is removed and rare gene is concentrated. However, such a method is not applicable unless the gene which is abundantly present is a known one.
On the other hand, the so-called subtraction method and modified methods thereof are available for the identification of mutant gene of some organism species (Ellen E. Lamer and E. Palmer (1984), Cell, 37, 171-177; Ilse Wieland, et al. (1990) Proc. Natl. Acad. Sci. USA, 87, 2720-2724; Anne Kallioniemi, et al. (1992) Science, 258, 818-821; and Nikolai Lisitsyn, et al. (1993) Science, 259, 946-951). They are the methods where, between the genes (say, A and B) in which most of them are the same in two individuals of the same organism species, gene which is different in terms of quality or quantity is concentrated and separated or where, in cDNA prepared from mRNA in the cells of the same type in two different states, genes (say, A and B) having different existing amounts are concentrated and separated. Its principle is that, when the genes in A(B) which are the same as B(A) are removed by genes of B(A) by any means, the specific gene which is present only in A(B) can be prepared. Accordingly, in the said method, two kinds of DNA samples in which most of the genes contained therein are the same are inevitably necessary.
However, in some specimens in the natural world (such as soil, lake water and river water), very many types of microbes are present and it is usual that the microbe composition varies for each specimen where each has its inherent composition. Accordingly, when rare gene derived from minor microbe therein is concentrated, it is impossible to prepare two kinds of DNA samples in which most of the genes contained therein—that which subtracts and is subtracted—are the same whereby concentration of the said rare gene using the above-mentioned subtraction method is not possible. Since rare gene in the cDNA sample is concentrated, it is also very difficult to prepare two kinds of cDNA samples wherein one cDNA sample contains the said rare gene and another does not contain the said rare gene where most of genes contained therein are the same. Consequently, it is difficult to concentrate the said rare gene by the above-mentioned subtraction method.